Telomerase elongates chromosome ends by addition of tandem telomeric repeats. This new DNA synthesis is required to balance the loss of DNA that is inherent in the incomplete replication of chromosome ends by conventional DNA polymerases. Single-celled eukaryotes constitutively activate telomerase and maintain a homeostasis of telomere length. Surprisingly, human somatic cells do not: they show progressive shortening of the telomeric repeat array with proliferation. Some human cells in the embryo, germline, epithelial tissues and hematopoietic system have detectable levels of telomerase catalytic activity in cell lysates, but this level of activation is insufficient to prevent an overall loss of telomere length in all human tissues with age. Cumulative loss eventually produces a repeat array that is too short to protect the chromosome end, resulting in a forced exit from the cell cycle. Cancer cells dramatically up-regulate telomerase to permit indefinite growth. For this reason, telomerase inhibitors have great promise as broadly effective anti-cancer therapeutics. Telomerase activators may have equally significant application for expanding the renewal capacity of normal somatic cells with critically short telomeres arising from genetics, disease, age, or environment. The telomerase RNA subunit (TER) is expressed as a precursor that must be processed, folded, and assembled as a stable ribonucleoprotein (RNP) complex in order to accumulate to detectable level in vivo. This RNP then recruits telomerase reverse transcriptase (TERT) to generate the active enzyme. Collins lab efforts in previous funding periods have contributed pioneering insights about the endogenous pathway of human TER processing and RNP biogenesis and discovered defects in telomerase RNP biogenesis that underlie X-linked and autosomal dominant forms of the bone marrow failure syndrome dyskeratosis congenita. The Specific Aims of the next funding period address crucial remaining gaps in knowledge about human telomerase RNP biogenesis and catalytic activation. Aim 1 exploits methods of transient and stable TER expression in human cells to discover and characterize additional RNA motifs and interacting proteins that direct telomerase RNP biogenesis. Aim 2 applies Collins lab expertise in RNA and protein affinity purification to elucidate the molecular mechanisms of human disease defects in telomerase RNP biogenesis. Aim 3 investigates telomerase RNP assembly with TERT to form the catalytically active enzyme. In vivo reconstitution methods will be combined with assays of catalytic activity in vitro and in vivo to elucidate the physiological specificity of human TER-TERT interaction and TER motif functions in the catalytic cycle of telomeric repeat synthesis. The long-term goal of these studies is to understand telomerase biogenesis, catalytic activation, and cellular regulation in normal cells and disease and to exploit this understanding for improvement of human health.